Endothelium lines blood vessels, and inter-connects all organ systems. There has been a growing appreciation that endothelial cells exhibit a rich diversity in structure and function. Such heterogeneity is apparent between endothelial cells in different organs, in endothelial cells along a single vascular segment within an organ and, indeed, between immediately adjacent cells. In the pulmonary circulation, endothelium in extra-alveolar blood vessels differs markedly from those in capillary segments. This program project grant is founded on the overall hypothesis that endothelium lining pulmonary arteries, capillaries, and veins is phenotypically distinct; each cell type is highly specialized to fulfill he unique demands of its vascular niche. We possess a limited understanding of how such heterogeneity is achieved to control site specific vascular demands, particularly in the lung's microvascular compartment. A principal goal in this amended renewal application is therefore to rigorously determine molecular mechanisms that allow lung microvascular endothelial cells to successfully control capillary function. Moreover, lung microvascular endothelial cell function is impaired by bacteria, such as Pseudomonas aeruginosa, during infection that culminates in acute lung injury. Hence, pulmonary microvascular endothelium represents a putative therapeutic target to combat vascular dysfunction in acute lung injury. The three projects in this amended renewal systematically study mechanisms regulating endothelial cell permeability and neutrophil transmigration; each project addresses three specific objectives, to: (1) identify and test novel molecular mechanisms (e.g. signatures) that control site-specific endothelial cell function, especially focusing on the microcirculation, (2) determine the importance of these mechanisms in preclinical models of disease, and (3) translate novel therapeutic approaches in preclinical models of disease. Projects are highly interactive both conceptually and pragmatically. This Program Project Grant draws on emerging developments in different fields of study, and applies these developments to generate new information about how microvascular endothelial cells, in particular, respond to inflammation and how they repair following injury. Defining the mechanisms that underlie lung microvascular endothelial cell function will provide insight into the site-specific nature of pulmonary vascular disease, and allow us to ultimately develop rational pharmacological therapies to discretely intervene in endothelial cell dysfunction that occurs in all known pulmonary vascular diseases.